LID scientists are collaborating with scientists from MedImmune under a CRADA to generate candidate vaccines against avian influenza viruses of each subtype. The vaccines were generated using plasmid based reverse genetics and each contains the hemagglutinin and neuraminidase genes from an avian influenza virus and six internal gene segments from the AA ca virus. In February 2013, a novel avian-origin H7N9 subtype influenza virus emerged in China causing severe lower respiratory tract disease in humans. A total of 135 human cases including 45 deaths occurred in the first wave from February to May 2013 (including 2 cases in July). From October 2013 a second wave of human infection began that caused more than 200 cases. We generated a live attenuated H7N9 influenza vaccine viruses that possesses the hemagglutinin (HA) and neuraminidase (NA) gene segments from the newly emerged wild-type (wt) A/Anhui/1/2013 (H7N9) and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (AA ca) by reverse genetics. The H7N9 ca vaccine virus was immunogenic in ferrets. A single dose of vaccine conferred complete protection of ferrets from homologous wt A/Anhui/1/2013 (H7N9) and near complete protection from heterologous wt A/Netherlands/219/2013 (H7N7) challenge infection. Therefore, this H7N9 LAIV candidate has been selected for vaccine manufacture and clinical evaluation to protect humans from wt H7N9 virus infection. H2 influenza viruses have not circulated in humans since 1968 and therefore a significant portion of the population would be susceptible to infection should H2 influenza viruses re-emerge. H2 influenza viruses continue to circulate in avian reservoirs worldwide and these reservoirs are a potential source from which these viruses could emerge. Three reassortant cold-adapted (ca) H2 pandemic influenza vaccine candidates with HA and NA genes derived from the wild-type A/Japan/305/1957 (H2N2) (Jap/57), A/mallard/6750/1978 (H2N2) (mal/78), or A/swine/MO/4296424/2006 (H2N3) (sw/06) viruses and the internal protein gene segments from the A/Ann Arbor/6/60 ca virus were generated by plasmid-based reverse genetics (Jap/57 ca, mal/78 ca and sw/06 ca, respectively). The vaccine candidates exhibited the in vitro phenotypes of temperature sensitivity and cold adaptation and were restricted in replication in the respiratory tract of ferrets. In mice and ferrets, the vaccines elicited neutralizing antibodies and conferred protection against homologous wild-type virus challenge. Of the three candidates, the sw/06 ca vaccine elicited cross-reactive antibodies and provided significant protection against the greatest number of heterologous viruses. These observations suggest that the sw/06 ca vaccine should be further evaluated in a clinical trial as a H2 pandemic influenza vaccine candidate. Live attenuated cold adapted (ca) H5N1, H7N3, H6N1 and H9N2 influenza vaccine viruses that were developed by LID and MedImmune replicated in the respiratory tract of mice and ferrets and 2 doses of vaccines were immunogenic and protected these animals from challenge infection with homologous and heterologous wild type (wt) viruses of the corresponding subtypes. However, when these vaccine candidates were evaluated in Phase I clinical trials, there were inconsistencies between the observations in animal models and humans. The vaccine viruses did not replicate well and immune responses were variable in humans, even though the study subjects were seronegative to the vaccine viruses before vaccination. Therefore, we sought a model that would better reflect the findings in humans and evaluated African green monkeys (AGMs) as a non-human primate model. The distribution of sialic acid receptors in the respiratory tract of AGMs was similar to humans. We evaluated the replication of wt and ca viruses of avian influenza (AI) subtypes, H5N1, H6N1, H7N3, and H9N2 in the respiratory tract of AGMs. All of the wt viruses replicated efficiently while replication of the ca vaccine viruses was restricted to the upper respiratory tract. We also evaluated the immunogenicity and protective efficacy of H5N1, H6N1, H7N3 and H9N2 ca vaccines. Protection from wt virus challenge correlated well with the level of serum neutralizing antibodies. Immune responses were slightly better when vaccine was delivered by both intranasal and intratracheal delivery than intranasally by sprayer. We conclude that live attenuated pandemic influenza virus vaccines replicate similarly in AGMs and human subjects, and that AGMs may be useful model to evaluate the replication of ca vaccine candidates. The influenza virus PB2 protein is a major determinant of the virus host-range. Lysine (K) at amino acid (aa) 627 of PB2 (PB2-627) is generally seen in viruses isolated from mammals while glutamic acid (E) is found in viruses from avian hosts. A pair of wild-type (wt) human H5N1 isolates, A/Vietnam/1203/04 (VN1203) with PB2-627K and A/Vietnam/1204/04 (VN1204) with PB2 627E isolated from pharyngeal swab and tracheal aspirate, respectively from the same patient differed by a K and E disparity at PB2-627. Direct sequencing of viral RNA from clinical material showed a mixed population, suggesting that the latter were independently selected during replication in the human respiratory tract, and are markers of mammalian adaptation. When and where the adaptive mutations in the PB2 protein occur remained unclear. The goal of our study was to determine when in the course of infection in a mammal and in which part of the respiratory tract the change at PB2-627 in a highly pathogenic avian influenza H5N1 virus would occur. Using deep sequencing, we defined the viral population from the upper and lower respiratory tracts of mice over the course of 7 days following intranasal administration of each virus. We found that a glutamic acid to lysine adaptive mutation at PB2-627 appeared synchronously in the upper and lower respiratory tract of infected mice and the transition was complete on day 6 post-infection. The adaptive mutation correlated well with efficient replication of the virus. Our data provides direct evidence of the importance of the PB2-627K mutation in the adaptation of avian viruses to mammals and identifies a critical window that should be targeted for antiviral treatment. Although lymphopenia is a hallmark of severe infection with highly pathogenic H5N1 and the newly emerged H7N9 influenza viruses in humans, the mechanism(s) by which lethal H5N1 viruses cause lymphopenia in mammalian hosts remains poorly understood. Because influenza-specific T cell responses are initiated in the lung draining lymph nodes, and lymphocytes subsequently traffic to the lungs or peripheral circulation, we compared the immune responses in the lung draining lymph nodes following infection with a lethal A/HK/483/97 or non-lethal A/HK/486/97 (H5N1) virus in a mouse model. We found that lethal H5N1, but not non-lethal H5N1 virus infection in mice enhances Fas ligand (FasL) expression on plasmacytoid dendritic cells (pDCs), resulting in apoptosis of influenza-specific CD8+ T cells via a Fas-FasL mediated pathway. We also found that pDCs, but not other DC subsets, preferentially accumulate in the lung draining lymph nodes of lethal H5N1 virus-infected mice and that the induction of FasL expression on pDCs correlates with high levels of IL-12p40 monomer/homodimer in the lung draining lymph nodes. Our data suggest that one of the mechanisms of lymphopenia associated with lethal H5N1 virus infection involves a deleterious role for pDCs in which elimination of influenza-specific CD8+ T cells by pDC-induced apoptosis contributes to lymphopenia in lethal HPAI H5N1 infection.